Such a microscope and/or microscopy method are or is known, for example, from the publication of C. Müller and J. Enderlein, Physical Review Letters, 104, 198101 (2010), or the EP 2317362 A1, which also cites additional references to the prior art.
This approach achieves an increase in resolution by imaging a spot in a diffraction limited manner onto a detection plane. The diffraction-limited image images a point spot as an Airy disk.
This diffraction disk is detected in the detection plane in such a way that its structure can be resolved. Based on the imaging performance of the microscope, the result is an oversampling on the part of the detector. When imaging a point spot, the form of the Airy disk is resolved. The resolution can be increased by a factor of 2 beyond this diffraction limit by suitably evaluating the diffraction pattern, which is described in the aforementioned steps and the disclosure of which is hereby incorporated in its entirety.
At the same time, however, it is unavoidable on the detection side that for each point, which is scanned on the sample in this manner, compared to a conventional laser scanning microscope (hereinafter also referred to by the acronym LSM), a single image has to be captured with a plethora of image data. If the structure of the single image of the spot is detected, for example, with 16 pixels, then each spot would have not only 16 times the amount of data, but also a single pixel would have an average of only 1/16 of the radiation intensity that would fall on the detector of an LSM during a conventional pinhole detection. Since the radiation intensity is, of course, not uniformly distributed throughout the structure of the single image, for example, the Airy disk, the radiation intensity at the edge of this structure is actually much less than the mean value of 1/n for n pixels.
Therefore, one is faced with the problem of the detector being able to achieve a high resolution detection of radiation quantities. Conventional CCD (charge coupled diodes) arrays, which are commonly used in microscopy, do not achieve a sufficient signal-to-noise ratio, so that even an extension of the image acquisition time, which by itself would already be a disadvantage in the application, would not help. APD (avalanche photodiode) arrays are also subject to excessive levels of dark noise, so that even an extension of the measuring time would result in an insufficient signal-to-noise ratio. The same applies to CMOS detectors, which are also disadvantageous with respect to the size of the detector element, because the diffraction-limited single image of the spot would fall on too few pixels. PMT (photo multiplier tube) arrays are also associated with similar design space problems. In this case the pixels are too large. Therefore, the design space problems are based, in particular, on the fact that a high resolution microscope can be realized in terms of the development effort and the distribution of the device, only if integration into existing LSM designs were possible. In such microscopes, however, specific sizes of the single image are specified. A detector, which is larger in terms of area, could be incorporated, only if it were possible to provide, in addition, an optical system that once again significantly expands the image by several orders of magnitude. Such an optical system is expensive and complicated in its design, if the objective is to obtain a diffraction-limited pattern without additional aberrations.
Other methods that avoid the above described problems associated with the high resolution detection are also known from the prior art. For example, the EP 1157297 B1 discloses a method that exploits nonlinear processes by means of structured illumination. A structured illumination is moved across the sample in a plurality of rotational and spatial positions; and the sample is imaged in these different states on a wide field detector, for which the described limitations do not exist.
A method, which also achieves a high resolution (i.e., a resolution of a sample image beyond the diffraction limit) without the described limitations of the detector, is known from the WO 2006127692 and the DE 10 2006021317. This method, which is known by the acronym PALM [Photo Activated Localization Microscopy], uses a marker substance that can be activated by means of an optical activation signal. Only when the marker is in the activated state is it possible for the marker substance to be excited with excitation radiation to emit a certain fluorescence radiation; even when exposed to excitation radiation, non-activated molecules do not emit any fluorescence radiation. Thus, the activation radiation switches the activating substance into a state, in which it can be excited to fluoresce. Therefore, one generally speaks of a switch-over signal. At this point this switch-over signal is applied in such a way that at least a certain proportion of the activated marker molecules are spaced apart from the adjacent marker molecules, which are also activated in such a way that the activated marker molecules, measured on the basis of the optical resolution of microscopy, are separated or can be subsequently separated. This procedure is referred to as isolating the activated molecules. For these isolated molecules it is easy to determine the center of their resolution-limited radiation distribution and, based thereon, to computationally determine the location of the molecules with higher accuracy than the optical imaging actually allows. In order to image the entire sample, the PALM method exploits the fact that the probability of a marker molecule being activated by the switch-over signal of a given intensity is the same for all marker molecules. Hence, the intensity of the switch-over signal is applied in such a way that the desired isolation occurs. These process steps are repeated until as many of the marker molecules as possible are included once in a subset that was excited to fluoresce.
Therefore, an object of the invention is to provide a microscope and/or a microscopy method, with which a high resolution can also be achieved. In particular, the objective is to enable fast image acquisition with high resolution microscopy.
The invention achieves this engineering object by means of a microscope of the type described in the introductory part, wherein the detector device has: a detector array, which has pixels and is larger than the single image, and a non-imaging redistribution element, which is disposed upstream of the detector array and which distributes the radiation from the detection plane in a non-imaging manner among the pixels of the detector array.
The invention achieves this engineering object by means of a method of the type, described in the introductory part, by providing a detector array that has pixels and is larger than the single image, and by redistributing the radiation of the single image from the detection plane in a non-imaging manner among the pixels of the detector array.
The detection plane is conjugate in relation to the plane of the spot in the sample and corresponds to the pinhole plane of a normal LSM.
In accordance with the invention, the spot, which is scanned onto the sample, is imaged into a detection plane in such a way that said spot is quiescent. Then the radiation from the detection plane is redistributed in a non-imaging manner and directed onto the detector array. In this case the term “non-imaging” is based on the single image that is present in the detection plane. It goes without saying that individual surface areas of this single image can be imaged according to the laws of imaging. However, in this respect it is certainly possible for the imaging optical system to be located between the detector array and the redistribution element. However, the single image, which is present in the detection plane, is not preserved as such during the redistribution.
The term “diffraction limited” is not to be limited to the diffraction limit according to the Abbe theory, but is also to covers cases, in which due to concrete shortcomings or limitations, the theoretical maximum is missed by 20%. Even then, the single image has a structure that is referred to herein as a diffraction pattern. This diffraction pattern is oversampled.
This principle makes it possible to use a detector array that does not fit in its size to the single image. The detector array is larger or smaller than the single image to be detected in at least one expansion. The concept “different geometric design” includes both a different expansion of the detector array as well as an arrangement with a different aspect ratio, based on the height and the width of the expansion of the single image in the detection plane. In addition, the pixels of the detector array can also be too large for the necessary resolution. At this point it is also allowed that the contour of the pixel arrangement of the detector array be basically different from the contour of the single image in the detection plane. Finally, the detector array has, according to the invention, a different size than the single image in the detection plane. The redistribution in the method or more specifically the redistribution element in the microscope makes it possible to select a detector array without having to take into consideration the dimensional limitations and the pixel size restrictions, caused by the single image and its size. In particular, a detector line may be used as the detector array.
The image of the sample is created from a plurality of single images in the conventional LSM manner by scanning the sample with the spot; each of these single images is assigned a different scanning location, i.e., a different scanning position.
The concept, according to the invention, can be carried out simultaneously in a parallelized form for a plurality of spots, a method that is known for laser scanning microscopy. Then a plurality of spots on the sample are sampled in a scanning manner, and the single images of the plurality of spots lie still next to one another in the plane of detection. Then they are redistributed by either a common redistribution element, which is sufficiently large in terms of area, or by a plurality of redistribution elements and are then directed to one or more correspondingly larger individual detector arrays.
The following description focuses, as an example, on scanning with a single point spot. However, this approach is not to be construed as a restriction, and the elucidated features and principles also apply mutatis mutandis to the parallel scanning of several point spots as well as to the use of a line spot. The latter is, of course, only diffraction limited at right angles to the line extension, so that the relevant features of this description apply then only to one direction (transversely to the line extension).